Ion imbalances are the predominant environmental stress that plants have to overcome. Plants must maintain a precise balance of cytosolic and extracellular ion concentrations despite widely varying soil conditions. Vacuolar ion transporters, including calcium (Ca2+) transporter, are key mediators of cytosolic ion concentrations. Intracellular Ca2+ release from vacuoles may also play a centra role in plant signal transduction events. Consequently, vacuolar ion transporters are components of signal transduction events and are necessary for general ion homeostasis during all aspects of growth and development. We have initiated a molecular and genetic approach to understand the processes of ion homeostasis and Ca2+ signal transduction in and around the plant vacuole. We have isolated two genes, designated CAX1 and CAX2 (CAlcium eXchangers 1 and 2), which represent the first vacuolar Ca2+ transporters isolated from plants. Biochemical measurements demonstrate that CAX1p is a Ca2+/H+ antiporter and CAX2p is a vacuolar heavy metal/H+ transporter with weak Ca2+ transport properties. The focus of this proposal is to determine the function and regulation of the CAX genes in plants. Three strategies to achieve these goals will be pursued: a)analysis of CAX expression, regulation and localization will establish their temporal and spatial expression; b)phenotypic analysis of CAX mutants will elucidate their function(s) in plants; and c)identification and characterization of plant CAX regulatory components. Once these transporters have been further characterized and additional regulators of ion homeostasis have been identified, then manipulating plant ion storage, signal transduction events, and altering the environmental constraints of agriculturally important plants, becomes realistic goals.